10. A process for making a food product comprising using a peptide derived
from a milk protein as an ice-structuring agent.

11. The process according to claim 10, wherein the milk protein is casein.

12. The process according to claim 10, wherein the peptide is derived by
cleaving the milk protein using a process selected from the group
consisting of chemical and enzymatic.

13. The process according to claim 10, wherein the peptide is obtained
through fractionation of a chemical or enzymatic cleavage substrate.

14. The process according to claim 10 comprising the step of producing a
frozen confectionery product.

15. Process for improving the heat shock resistance of a frozen
confectionery product comprising the steps of:cleaving a milk protein
into peptides;isolating the peptides so obtained;using the peptides to
manufacture a frozen confectionery product.

16. Process of claim 15, comprising performing a fractionation step on the
peptides.

17. Process according to claim 15, wherein the milk protein is casein.

18. Process according to claim 15, wherein the cleavage step is selected
from the group consisting of enzymatic and chemical.

19. Process according to claim 18, wherein the enzymatic cleavage is
carried out with an enzyme selected from the group consisting of trypsin,
papain, neutrase and mixtures thereof.

20. Process according to claim 18, wherein the enzymatic cleavage is
performed for a period of 5 to 480 min.

21. Process according to claim 18, wherein the enzymatic cleavage is
performed at a temperature between 45.degree. C. and 70.degree. C.

22. Process according to claim 18, wherein the enzymatic cleavage is
performed at a pH between 6.5 and 8.5.

23. Ice-structuring peptide obtainable by an enzymatic cleavage of casein.

24. Peptide according to claim 23, wherein the cleavage of casein is
performed using an enzyme selected from the group consisting of trypsin,
papain, neutrase and mixtures thereof.

25. Peptide according to claim 23, wherein the cleavage is performed for 5
to 480 minutes, at a temperature between 45.degree. C.-70.degree. C. and
a pH between 6.5 and 8.5.

Description:

FIELD OF THE INVENTION

[0001]The present invention relates to the use of peptides of lactic
origin as ice-structuring agents, to their use in the manufacture of
frozen products, and to the frozen products comprising them.

BACKGROUND OF THE INVENTION

[0002]Ice-structuring proteins (ISPs) or abusively named anti-freeze
proteins (AFPs) naturally occur in a range of species that are
susceptible to freeze damage, i.e. to species that are found in sub-zero
environments. They have evolved in nature to help many different
organisms e.g. fish, insects, plants and bacteria, to survive in these
cold environments. To date, fish from cold climates has been considered
as the main source of ISPs (cf. for example WO 9702343 (Unilever), EP
0788745 (Nestle)). Plant sources of ISPs are also described in for
instance EP 0959689 (Unilever), EP 0918863 B1 (Unilever), EP 1049713
(Unilever), EP 1049783 B1 (Unilever), EP 1276763 (Unilever). Further, EP
1240188 (Unilever) discloses ISPs isolated from bacterial sources from
low-temperature environments. WO 9804147 (Unilever) reports the isolation
of peptides that inhibit ice-crystal growth, derived from plants such as
rye or grass.

[0003]It is thought that ISPs achieve their function by binding to
specific planes of the ice crystals and by minimising recrystallisation
(Biophysical Journal, February 1991, 409-418). Inhibition of ice
recrystallisation, also referred to as ice crystal growth suppression
(Cryobiology, 25, 55-60, 1988) is a property of ISPs that can be tested
by comparing at a certain point in time the ice crystals in the presence
of ISP and in the absence of ISP. The application of this method in the
testing of fish ISPs is described in U.S. Pat. No. 5,118,792 (DNA Plant
technology Corporation). Thus, it has been reported that ISPs from
Antarctic fish are very effective in minimizing ice crystal growth
(Cryobiology 32:23-34, 1995). Compared to fish ISPs, an ISP found
recently in rye grass (Lolium perenne) is reported to be even 200 times
more effective (in molar terms) in inhibiting recrystallisation (Nature,
406(6793):256, 2000).

[0004]Another property of ISPs is their ability to influence the shape of
ice crystals. This property stems from selective binding of ISPs to
certain faces of the ice crystals and therewith limiting crystal growth
in certain directions. The presence of ice crystals having a hexagonal
bipyramid shape is then considered indicative of the presence of ISP.
This method is described for testing the activity of extracellular winter
rye ISPs in WO 92/22581 (University of Waterloo).

[0005]ISPs also have the ability to inhibit the activity of ice nucleating
substances. This interaction between an ISP and ice nucleator may for
example result in increasing thermal hysteresis (WO 96/40973--University
of Notre Dame du Lac). Thermal hysteresis is characterised by a lowering
of the apparent freezing temperature of a solution without affecting the
melting temperature. Thus, the identification of sources of ISPs by
thermal hysteresis tests is widely described in the literature (e.g. John
G. Duman, Cryobiology, 30, 322-328, 1993).

[0006]It has been suggested that it is the tertiary structure of these
proteins which allows them to interact with ice (cf. Fletcher et al.,
Annu. Rev. Physiol., 2001, 63:359-390). Thus, all these properties render
ISPs applicable to a range of potential uses. Most importantly, ISPs have
been suggested for improving the freezing tolerance of products. Frozen
products can be subjected to temperature fluctuations leading to an
increase in ice crystal size and thus to textural defects. ISPs thus
enable frozen products to withstand temperature fluctuations that may
occur during packaging, storing, manufacturing etc., thus allowing them
to keep a desirable texture.

[0008]However, sources of ISPs have been limited to sources from sub-zero
environments and/or to the use of genetically modified organisms (GMOs)
for producing these proteins. For instance, WO 9403617 (Unilever)
discloses the production of ISPs from yeast and their possible use in ice
cream. WO 9611586 (HSC R&D Ltd--Seabright Corporation Ltd) describes fish
ISPs produced by microbes. Others ISPs have mainly been obtained by
enzymatic and chemical modification. For example, WO 9013571 (DNA Plant
technology Corporation) discloses ISP peptides produced chemically or by
recombinant DNA techniques from plants.

[0009]These techniques are subject to much controversy and the resulting
"GMO labelled" products are not always appealing to the consumer.

OBJECT OF THE INVENTION

[0010]It is thus an object of the invention to provide an alternative
source of ice-structuring agent which can be used in frozen products and
which avoids the need to use genetically manipulated additives.

SUMMARY OF THE INVENTION

[0011]Accordingly, this object is solved by the features of the
independent claims. The dependent claims further develop the central idea
of the invention.

[0012]Thus, in a first aspect, the invention provides a frozen food
product comprising at least one ice-structuring peptide derived from milk
protein.

[0013]In a second aspect, the invention relates to the use of a peptide
derived from milk protein as ice-structuring agent.

[0014]A process for improving the heat shock resistance of frozen
confectionery product comprising the steps of: [0015]a. Cleaving a milk
protein into peptides [0016]b. Isolating the peptides obtained in the
previous step and [0017]c. Using said peptides in the manufacture of a
frozen confectionery productalso forms part of the invention.

[0018]Finally, the present invention also encompasses an ice-structuring
peptide obtainable by enzymatic cleavage of casein.

FIGURES

[0019]The present invention is described hereinafter with reference to
some of its embodiments (or reference embodiment) shown in the figures,
wherein

[0023]The present invention relates to frozen food products which comprise
at least one ice-structuring peptide. By "peptide" is meant a chain of up
to 50 amino acids linked by peptide bonds. The peptides of the invention
are not proteins and do not comprise a tertiary structure. They are
protein hydrolysates. The weight of said peptide is less than 1 kDa. By
"ice-structuring" is meant that the peptide is able to interact at the
ice crystal interface, in particular to inhibit ice crystal growth. This
function is in contrast to and distinguishable from the aerating/air cell
stabilising function of some peptides which occurs at the air cell
interfaces.

[0024]The peptides used in the present invention are derived from milk
protein. Milk proteins include casein and whey protein. Preferably, the
milk protein used is casein. By "casein" is meant casein as it is found
naturally, i.e. casein which has not been modified chemically. According
to the invention, this definition includes casein as such and
water-soluble caseinates, for example alkali metal, alkaline earth metal
and ammonium caseinates.

[0025]The peptide may be derived by enzymatic or chemical cleavage of said
milk protein. In a preferred embodiment, the milk protein is treated with
an enzyme, which may be selected from any protease enzyme capable of
hydrolysing the milk protein into peptides. More preferably, the enzyme
is selected from trypsin, papain, neutrase or mixtures thereof. According
to the invention, upon treatment of milk protein with an enzyme, the
protein is cleaved into peptides which may be used as ice-structuring
agents. These are capable of controlling frozen food stability by
inhibiting crystal growth, thus improving the quality of the product.

[0026]Alternatively, it has been found that commercially available milk
protein hydrolysates may be used in the present invention. It is thus
thought that said commercial hydrolysates may comprise peptides according
to the invention, i.e. peptides which may be used as ice-structuring
agents. Such hydrolysates are for example sold under the name of
Peptigen, Peptone, Peptopro etc.

[0027]Crystal growth can be measured by crystal size analysis using a
computer-controlled image analyser. The size of crystals is usually
measured by the diameter distribution over the volume (i.e. over the
amount of ice crystals evaluated). Thus a Dv(0.50) represents the value
of the maximum diameter of 50% of the total number of ice crystals
evaluated. Dv(0.90) represents the value of the maximum diameter of 90%
of the total number of ice crystals evaluated. By using the peptides of
the invention, the crystal size of a sugar solution at -11° C.
containing said peptides is reduced by 15%-25% compared to a reference
sugar solution at -11° C. with no peptides (cf. Table 2).

[0028]The effect of the peptides is further evidenced by heat shock
treatment. By "heat shock" is meant the inevitable temperature cycling
during storage and distribution that creates ice crystals growth and
other deterioration due to structural change. This "heat shock" is
reproduced by a process, which is a defined cycle of thermal changes
inflicted on the product. The product is placed inside a cabinet set at
-20° C., which is automatically switched on for 19 hours and then
switched off for 5 hours using a 24-hour timer in order to provoke a
thermal shock.

[0029]These analyses led to the surprising conclusion that certain
casein-derived peptides have a visible and measurable action on the
inhibition of ice-crystal growth.

[0030]Indeed, referring to FIGS. 1, 2 and 3a and 3b, it can be seen that
although the crystal size in ice cream grows, in particular after 2 weeks
of heat shock, by using the ice-structuring peptides according to the
invention a smaller crystal size is obtained.

[0031]The manufacturing conditions of some of said peptides which provide
anti-freeze activity are given in table 1.

[0032]Thus, the present invention relates to frozen food product of the
invention which comprise the peptide of the invention in an amount
between 0.0001-10%, preferably in an amount between 0.001-5%, more
preferably in an amount between 0.01-1% by weight of the composition.

[0033]The frozen food product of the invention may be any food product.
Preferably, it is a food confectionery product which may be ice cream,
water ice, sorbet, frozen yogurt, mellorine etc. The product may comprise
inclusions in the form of chocolate pieces, nuts, pieces of fruits etc.
The product may also comprise a coating, such as e.g. a chocolate coating
or a fruit coating etc. The coating itself may also contain inclusions.
The frozen food product may be aerated or non-aerated. Aerated frozen
confections preferably have an overrun of from 30% to 200%, more
preferably from 50% to 150%. For example, the level of overrun in ice
cream is typically from about 70% to 100%, and in confectionery such as
mousses the overrun can be as high as 200 to 250 wt %, whereas the
overrun in milk ices is from 25 to 35%.

[0034]Thus, the invention encompasses the use of a peptide derived from
milk protein as ice-structuring agent. The milk protein is preferably
casein and the peptide may be derived from the milk protein by chemical
or enzymatic cleavage of the protein.

[0035]According to another embodiment of the invention, the peptide may be
obtained through fractionation of the substrate obtained by chemical or
enzymatic cleavage of the protein. This provides the advantage of
enriching the active principle and thus to work at lower concentrations.

[0036]According to the invention, the ice-structuring peptide is
preferably used in frozen confectionery products.

[0037]Referring to FIG. 3b, it can be seen that by using the peptides
according to the invention, the ice crystals are smaller compared to a
standard ice cream mix (FIG. 3a).

[0038]Furthermore, it has been observed that the shape of ice crystals in
ice cream maintains an essentially round aspect. This effect is
surprising in view of the fact that regular anti-freeze proteins used in
the art tend to modify the structure of ice crystals. Indeed, ice
crystals found in frozen products containing ISPs tend to have an
elongated, rectangular shape which affects the texture of the product by
increasing its hardness. By the present invention, a frozen product
having a smooth, soft texture may be obtained while still being resistant
to temperature fluctuations.

[0039]In a further aspect, the invention thus provides a method for
improving the heat shock resistance of frozen confectionery products.

[0040]The first step in the method consists in the cleavage of the milk
protein. Preferably the milk protein is casein. Cleavage may be carried
out chemically or enzymatically. A preferred process is enzymatic
hydrolysis of the milk protein in order to yield peptides. For the
hydrolysis of milk protein, the enzyme/substrate ratio is from 1/100 to
1/500, and preferably 1/250 w/w.

[0041]The peptides obtained may vary widely depending on the conditions
used e.g. incubation temperature, incubation time, the pH of the solution
etc. According to the invention, it has been found that an incubation
temperature between 45° C.-70° C., an incubation time
between 5 and 480 minutes and a pH of the solution between 6.5 and 8.5
are preferred conditions in order to obtain different peptides which are
all efficient ice-structuring compounds.

[0042]The enzyme used may be selected from any protease enzyme.
Preferably, it is selected from trypsin, papain, neutrase or mixtures
thereof.

[0043]Thus, the milk protein is incubated with the desired enzyme under
determined conditions. After the desired period of time, the enzyme is
then inactivated and the substrate is collected. Peptides are then
isolated from the substrate and may be used directly in the production of
a frozen product. Alternatively, the substrate may further be subjected
to fractionation, after which the peptides are isolated and used as
ice-structuring agents in the manufacture of a frozen product. The
peptides may also be further purified prior to use. The frozen product
may be manufactured by any method known to the skilled person. Further,
the peptides of the invention may be added at any stage during
manufacture of the frozen product, more preferably during mix
preparation, before maturation time.

[0044]The invention thus also relates to ice-structuring peptides
obtainable by enzymatic cleavage of casein. The enzyme cleavage may be
carried out by any embodiment of a process described above.

[0045]In summary, the present invention provides a way to produce frozen
products, and in particular frozen confectionery products which are
smooth and stable after heat shock. It also provides for natural frozen
products which have a "clean" label and are free of GMO additives.
Furthermore, the modification of the ice crystal structure observed when
using known ISPs is no longer observed. This yields a product which
retains essentially circular ice crystals and maintains a smooth,
palatable texture after heat shock.

[0046]The present invention is further illustrated by the following
non-limiting examples.

Examples

Example 1

Manufacture of Peptides

[0047]Commercial sodium caseinates were subjected to the action of
different enzymes and the resulting hydrolysates were tested for their
inhibitory activity on ice-crystal growth.

[0048]The hydrolysis is carried out according to the following procedure.
The sodium caseinate protein isolate is dissolved at a concentration of
5% of proteins in water. The pH is adjusted to the desired pH according
to table 1 by adding either sodium hydroxide, 1N NaOH, or hydrochloric
acid, 1N HCl. The substrate is then brought to the desired temperature.
Finally, the enzyme is added. During reaction, the pH is not adjusted.

[0049]For the hydrolysis of the caseinate, the enzyme/substrate ratio is
from 1/100 to 1/500, and preferably 1/250 weight for weight.

[0050]Depending on the reaction conditions, the reaction time can go from
5 min up to 1200 min.

Example 2

Sample Preparation for Analysis

[0051]50 μl of the reaction product (obtained by the method described
in example 1) are mixed with RP-HPLC buffer for chromatographic analyses.
Simultaneously, 1.5 ml of the reaction product are heated at 90°
C. for 15 minutes with the aim of inactivating the enzymes. After
heating, the sample is centrifuged for 15 minutes at 14 000
revolutions/min (rpm), in order to eliminate any possible precipitates.
The supernatant is lyophilized for the analysis of ice-crystal
recrystallization.

Example 3

HPLC Analysis

[0052]The reverse-phase HPLC analysis is carried out on the samples
obtained in example 2 according to the method described in S. Visser, C.
J. Stangen and H. S. Rollema (1991) "Phenotyping of bovine milk proteins
by reversed-phase high-performance liquid chromatography", J.
Chromatography, 548, pp. 361-370. The separation is based mainly on the
differences in hydrophobicity of the proteins and of the peptides. The
detection is carried out by UV absorption at 22 nm.

Example 4

Ice Recrystallisation Test

[0053]To evaluate the effect of the lactic protein hydrolysates on
ice-crystal recrystallization, an analysis of ice-crystal
recrystallization is carried out.

[0070]The lyophilized hydrolysate obtained according to the procedure
described above is dissolved in a 40% solution of sucrose in water. The
final solution contains 5% by weight of lyophilized hydrolysate. A 40%
solution of sucrose in water is used as a reference sample. A solution of
peptides that inhibit ice-crystal growth (ISP type 1) in a 40% solution
of sucrose in water is used as a positive control. The samples are
analysed by observation under a microscope of Polyvar type sold by
Reichert-Jung, Harnalser Hauptstrasse 219, Vienna, Austria, equipped with
a Linkham temperature regulator sold by Linkham Scientific Instruments
Ltd, Tadworth UK. The temperature regulator is pre-calibrated with
n-dodecane (melting point: -9.6° C.) and n-decane (melting point:
-29.7° C.)

[0071]A 2 μl sample is placed on a quartz cell covered with a circular
cap. The quartz cell is placed on the temperature regulator and then
cooled to -100° C. at a rate of 90° C. per minute. At
-100° C., the sample is left to equilibrate for 2 minutes, and
then reheated to -11° C. at a rate of 30° C. per minute.
The time zero of the analysis is taken at the instant the sample reaches
-11° C. During the first two minutes of the analysis, the
microscope is regulated in order to ensure a good image and sufficient
crystals for a significant analysis. After 2 minutes, the images from the
microscope are acquired and stored using a video recorder software with a
pre-defined time lapse (2 minutes). The images are recorded for each
hydrolysate for 2 h at constant temperature. Results are shown in table
2.

[0072]For each sample taken, the 40% sucrose solution is taken as a
reference. For this solution, the ice crystals reach the average maximum
size that can be reached for a given cooling/heating cycle since no
ice-crystal growth inhibitor is present. The results obtained for the
various hydrolysates can thus be compared with the microscope images of
the ice crystals in the reference sucrose sample. By comparing the
microscope images of the state of the crystals after one hour at
-11° C. with the image obtained for the reference sucrose
solution, it is possible to establish whether or not an ice-crystal
growth inhibition effect is observed for each hydrolysate tested.

[0073]Micrographs show the evolution of ice crystal during a typical
experiment using the conditions described herein.

[0075]The results of this analysis confirm that the size of the ice
crystals obtained is smaller in the five solutions containing the casein
hydrolysates selected than in the 40% sucrose reference solution without
agent for inhibiting ice-crystal growth. This shows the inhibitory effect
on ice-crystal growth of these five hydrolysates.

Example 3

Ice Cream Recipes Used for Trials

[0076]The following recipes were used in the trials at 1, 5 and 10% in ice
cream. The standard mixes with an equivalent total solids (TS) content
were used as reference.

[0078]Technical tasting carried out after 3 weeks of heat shock found that
the standard mix was very crystallised in the mouth. The perception of
ice crystals after heat shock was lower in ice cream samples containing
the peptides of the invention.